The long-term objective of this research proposal is to investigate the mechanism(s) by which SOD2 gene expression is altered during differentiation and carcinogenesis with a goal to determine molecular targets for therapeutic intervention that could be used to reactivate this important antioxidant enzyme and tumor suppressor gene. The hypothesis for the proposed research is that SOD2 gene expression is regulated, at least in part, through an epigenetic mechanism involving DNA methylation and/or histone modification of its transcriptional regulatory regions. To test the hypothesis that the human SOD2 gene is regulated by epigenetic events including DNA methylation, histone hypoacetylation, and chromatin accessibility, we propose to pursue the following specific aims: 1) determine whether decreased expression of the SOD2 gene in human cancer cells is causally associated with hypermethylation of the CpG island in the human SOD2 gene; 2) determine whether and to what extent histone modifications including acetylation and/or methylation participate in the transcriptional inhibition of the SOD2 gene in human cancer cells; 3) test the hypothesis that the known regulatory regions of the endogenous SOD2 reside in a closed heterochromatic state in non-expressing human cancer cells; 4) establish the molecular mechanism(s) by which the transcription factor AP-2 leads to the down-regulation of SOD2 expression in human cancer cells; and 5) examine the relationship between p53 and SOD2 expression and to determine the mechanism(s) underlying p53 mediated repression of SOD2 expression. The list of important cancer genes, particularly tumor suppressor genes, whose transcriptional regulation during cancer development is governed at least in part by epigenetic mechanisms including aberrant cytosine methylation, histone hypoacetylation and heterochromatinization makes this an important area of investigation. The proposed studies will not only clarify the specific mechanism(s) by which these epigenetic factors participate in SOD2 regulation, but also provide valuable insight into new avenues for clinical intervention and treatment. As our understanding of epigenetic control of gene expression continues to expand, novel therapeutic approaches targeting this level of gene regulation will undoubtedly emerge. The acknowledged importance of decreased SOD2 expression in cancer makes this a particularly attractive target gene for therapeutic manipulation by pharmacological inhibitors of DNA methyltransferases and histone deacetylases.